PCT-65 serotonin receptor

ABSTRACT

The PCT-65 serotonin receptor gene was cloned and characterized from rat and human libraries. This receptor has nucleotide and amino acid homology with previously described 5-HT genes and can bind ligands that are known to interact with serotonin receptors.

This application claims priority from PCT Application No. PCT/US93/10301filed on Oct. 26, 1993. This application is also a continuation-in-partof U.S. patent application Ser. No. 07/980,514, filed on Oct. 26, 1992,now abandoned.

FIELD OF THE INVENTION

This invention relates to cloning and characterization of cellularreceptors. Specifically, this invention relates to the cloning andcharacterization of the PCT-65 serotonin receptor protein.

BACKGROUND OF THE INVENTION

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has a varietyof functions in the central nervous system. It has been implicated inmany cognitive and behavioral functions, including aggression, sexualbehavior, learning and sleep. Disruptions of serotonergic systems may bea critical factor in a number of clinical disorders or conditionsincluding schizophrenia, depression, obsessive compulsive disorder,anxiety, migraine headaches, and pain.

The multitude of effects produced by serotonin are mediated by variousserotonin receptors which exist in the central and peripheral nervoussystem. The transduction of serotonergic signals across the neuronalmembrane is believed to be mediated by a diversity of receptor subtypeswhich, in mammals, appear to fall into four pharmacologically distinctclasses designated 5-HT₁ -5-HT₄. The 5HT₁ subcategory has been furthersubdivided into five different subtypes referred to as 5HT_(1A-E). Theprimary structures for a number of these receptors have been elucidatedby molecular cloning, including the 5-HT₁, 5-HT₂ and 5HT₃ subclasses. Inaddition, the sequences of three different Drosophila serotoninreceptors, 5-HT_(dro1) and 5-HT_(dro2A),B, have been reported.

Selective therapeutic agents, including agonist and antagonist drugs,have been developed based on serotonin receptor technologies utilizingthe serotonin classes known in the art. 5-HT₂ antagonists, for example,are useful in the treatment of schizophrenia, parkinsonism, and anxietydisorders. Several azapirones, such as buspirone, gepirone, andipsapirone, have high affinities for 5HT_(1A) receptors in the brain,and are useful in the treatment of anxiety. Highly selective 5-HT uptakeinhibitors, which have minimal effects on norepinephrine or dopamineuptake or on other neurotransmitter receptors, have been used tosuccessfully treat depression.

Characterization of proteins with serotonin receptor activity wouldclarify the role of serotonin in the central nervous system. Analysis ofthe receptor proteins and their functional role in the central nervoussystem would help elucidate the pathophysiological basis of many humandiseases. Accordingly, disclosed herein is a structurally andpharmacologically novel serotonin receptor which is distinct from anyclass of previously described 5-HT receptors.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B are photographs of Northern blots of rat Poly (A)⁺ RNAidentifying PCT-65 mRNA from various central nervous system (CNS) andperipheral tissues. RNA size markers (in kilobases) is provided at theright of each blot.

FIG. 2 illustrates the cloning procedure used to derive the PCT-65 clonewithout an intron for expression studies.

FIG. 3a-1 provides the results of binding studies to assess the abilityof ³ H!-LSD to bind to the surface of COS-7 cells transfected with aPCT-65 containing construct. FIG. 3a-2 additionally includes a Scatchardanalysis of the saturation data. FIG. 3b illustrates the pharmacologicalspecificity of ³ H!-LSD binding to transfected COS-7 cells in thepresence of various serotonin agonists and antagonists as well as theeffect of PCT-65 receptor modulation using Gpp(NH)p.

SUMMARY OF THE INVENTION

One embodiment of the present invention is the isolated mammalianserotonin receptor protein PCT-65. Preferably this receptor protein ishuman. The present invention also encompasses species variations of thePCT-65 receptor.

Another embodiment of the present invention is a method for screening adrug candidate for central nervous system activity by contacting thedrug candidate with the PCT-65 protein and measuring binding of the drugcandidate by the protein.

An additional embodiment of the present invention is a method forscreening drug candidate for central nervous system activity by firstcontacting the PCT-65 serotonin receptor protein with a first moleculeknown to be bound by the protein to form a first complex of the proteinand the first molecule; then contacting the first complex with the drugcandidate; and finally measuring whether the drug candidate displacesthe first molecule from the first complex. This method can preferablyinclude in the measuring step, the step of measuring the formation of asecond complex of the protein and the drug candidate. An alternativemethod of measuring the displacement of the first molecule can beaccomplished by measuring the amount of the first molecule that is notbound to the protein.

A further embodiment of the present invention includes a recombinantconstruct of the polynucleotide, preferably the polynucleotide of SEQ IDNO: 6 encoding the PCT-65 serotonin receptor protein operably linked toa heterologous promoter.

Another embodiment of the present invention is directed at thepolynucleotide sequence encoding the PCT-65 receptor. Preferably, thispolynucleotide sequence is Sequence ID NO: 6 or species variationsthereof. Additionally, the present invention encompasses an isolatednucleotide sequence having homology to at least 18 contiguousnucleotides of the PCT-65 gene.

The present invention also embodies a mammalian cell line, preferablyhuman, in continuous culture expressing the PCT-65 serotonin receptorprotein. Particularly, the cell line can contain the polynucleotide ofSequence ID NO: 6 or species variations thereof. One especiallypreferred embodiment includes HEK 293 immortalized cells in continuousculture expressing the PCT-65 receptor.

Yet another embodiment of the present invention is isolated antibodiesagainst the PCT-65 serotonin receptor protein. Preferably these isolatedantibodies are polyclonal; even more preferably, the isolated antibodiesare monoclonal.

An even further embodiment of the present invention includes a method ofscreening a ligand for binding to the PCT-65 serotonin receptor by thesteps of:

transfecting a cell line with a gene encoding the PCT-65 serotoninreceptor protein in an expression vector;

culturing the cell line to express the gene in media containing a ligandof the receptor; and

measuring the binding of the ligand the receptor produced by the cellline.

Preferably, in this method, the cell line is from a mammal, morepreferably a human, and most preferably the cell line is HEK 293. Anadditional preferred embodiment of the above method entails transfectingcells with the PCT-65 serotonin receptor encoded by the polynucleotideof SEQ ID NO: 6. Additional preferred embodiments of this method includeusing eukaryotic expression vectors, preferably pSRα-65. Anotherpreferred embodiment includes using ligands that are labeled with thelabel being either radioactive or colorimetric.

Still another embodiment of the present invention is a method ofdetermining the ability of a drug to inhibit ligand binding to thePCT-65 serotonin receptor protein by the following steps:

transfecting a cell line with a DNA sequence encoding PCT-65 in anexpression vector;

culturing the cell line to express the PCT-65 receptor in mediacontaining a ligand of the receptor;

determining the level of binding of the ligand to the receptor;

culturing the same cell line to express the receptor in the presence ofboth the ligand and the drug; and

determining the level of binding of the ligand to the expressedreceptor, wherein a lower level of binding in the presence of the drugindicates that the compound is an inhibitor of ligand binding.

Preferably, the cell line of this method is mammalian, most preferablyHEK 293 , and the ligand, the drug or both are labeled. Additionally,the expression vector of this method can preferably be pSRα-65 and theligand can advantageously be serotonin.

Still another embodiment of the present invention is a method ofdetermining the ability of a drug to inhibit ligand binding to thePCT-65 serotonin receptor protein by the steps of:

transfecting a cell line with a DNA sequence encoding PCT-65 in anexpression vector;

culturing the cell line to express the PCT-65 receptor in mediacontaining a ligand of the receptor;

isolating the membranes from the cell line, wherein the membranescontain the expressed PCT-65 receptor;

determining the level of binding of the ligand to the receptor on themembranes;

incubating the membranes in the presence of both the ligand and thedrug; and

determining the level of binding of the ligand to the membranes, whereina lower level of binding in the presence of the drug indicates that thecompound is an inhibitor of ligand binding.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses the identification of a novel serotoninreceptor, PCT-65 , having both structure and pharmacological propertiesdistinct from any class of previously described 5-HT receptors.

The gene sequence of PCT-65, vectors encoding this sequence, cellsexpressing PCT-65 from vectors encoding this sequence and substantiallypurified protein encoded by this sequence will be particularly usefulfor studying the cellular distribution of the protein. Additionally,assays using the expressed protein will be important tools for studyingthe pharmacological effects of serotonin receptor binding and blockingdrugs. Knowledge of the PCT-65 gene sequence will also allowintroduction of mutations into the protein to study protein function andreceptor binding activity, and ultimately to the development andidentification of agonists and antagonists with useful therapeuticpotential.

This receptor appears to be localized predominantly in the hippocampusand hypothalamus of the central nervous system in addition to otherlimbic and cortical regions. Lower levels of this receptor are alsofound in the periphery including the spleen and kidney. When expressedin mammalian cells, this receptor exhibits high affinity for serotoninas well as a number of tricyclic antipsychotic and antidepressant drugsincluding clozapine, loxipine, and amitriptyline. The pharmacologicalproperties that PCT-65 exhibits for these drugs, however, is differentfrom any other known 5-HT gene. Thus, PCT-65 represents an unknown anduncharacterized receptor protein.

The present invention includes isolated PCT-65 serotonin receptors fromrats, humans, other mammals, and other vertebrates. Furtherinvestigations of PCT-65's role in vivo and in vitro will help improvecurrent therapies for several neuropsychiatric disorders by providingadditional information on each drug's receptor mediated action.

While this gene sequence was originally isolated from rat kidneyproximal convoluted tubule, and has been found to be expressed invarious brain regions, it is anticipated that homologous proteins aresimilarly located in corresponding tissues from other vertebrates.Further, the methods disclosed herein can be used to identify proteinfrom other vertebrates having similar pharmacologic properties. Inaddition, nucleotide and protein sequences of greater than or equal to90% homology from other vertebrates can readily be located using thetechniques of this invention.

IDENTIFICATION OF NOVEL MEMBERS OF THE G PROTEIN-COUPLED RECEPTOR FAMILY

As explained below in Example 1, degenerate polymerase chain reaction(PCR) primers SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized by standardmethods and modeled after sequences from the third and sixthtransmembrane (TM) regions of previously cloned biogenic aminereceptors.

The primers were used to amplify cDNA synthesized from poly (A)+ RNApurified from rat kidney proximal convoluted tubule cells. PCRamplification with the degenerate primers yielded a number of differentcDNA fragments, some of which were determined by DNA sequence analysisto represent portions of previously cloned receptors. In addition, someputatively novel receptor DNA sequences, as well as unknown DNAsequences, were identified using this method. One putatively novelreceptor cDNA fragment, termed PCT-65₁, exhibited high homology withpreviously cloned members of the serotonin receptor family and wasselected for further study.

CHARACTERIZATION OF THE PCT-65 CLONE

The tissue distribution of the corresponding mRNA was examined byNorthern blot as an initial characterization of the PCT-65₁ clone, FIGS.1A, 1B. To determine the tissue distribution of the clone, poly(A⁺) RNAwas prepared from a variety of vertebrate tissues. For example, ratswere sacrificed and the tissue of interest was dissected from theanimal. To preserve the integrity of the mRNA, the intact tissue samplewas either flash frozen in liquid nitrogen or homogenized directly inguanidine isothiocyanate.

FIGS. 1A, 1B and photographs showing Northern blots of poly (A+) RNAprepared from a variety of rat tissues associated with the centralnervous system as well as selected peripheral tissues. Techniques forobtaining poly (A⁺) mRNA from tissue homogenates are well known in theart. Additionally, commercial kits are available for the preparation ofpoly (A⁺) mRNA. Examples of commercially available kits are theFastTrack™ kits from Invitrogen (San Diego, Calif.) or those availablefrom CINNA/BIOTECK, Laboratories International.

The Northern blots of FIG. 1 were probed with the isolated PCT-65₁fragment (generated in Example 1) corresponding to transmembrane regionsIII and IV. The fragment was random primed using commercially availablekits, such as those available from Stratagene, Inc. (San Diego, Calif.).The preferred methods for degenerate PCR construction and random primingare provided in Example 1.

Example 2 discloses a preferred Northern blotting procedure useful foridentifying mRNA transcripts binding to the PCT-65₁ fragment. In thisexperiment, a single transcript of ˜3.6 kb was observed in varioushomogenates from the central nervous system. The highest level of mRNAexpression occurred in the hippocampus, however identifiable signalswere present in the olfactory tubercle (OFT), mesencephalon (MES),hypothalamus (HYP), hippocampus (HIP) and cortex (CTX). The olfactorybulb (OFB) had reduced levels while the cerebellum (CB), the retina(REN) and the total brain homogenates (TB) had levels of mRNA that werevery reduced.

In peripheral tissues, this mRNA transcript was identified most heavilyin the spleen, whereas it was present in undetectable levels in thepituitary (PIT), testis (TEST), stomach (STOM), prostate (PROS), ovary(OV), skeletal muscle (SKM), lung (LNG), liver (LIV), kidney (KID) andgut (GUT). While kidney is the tissue of origin of the PCT-65 clone, theinability to detect PCT-65 mRNA in kidney via Northern blotting suggeststhat PCT-65 is either a minor transcript in the kidney or is restrictedto a small population of cells.

ISOLATION OF A FULL LENGTH PCT-65 CLONE

Based on the high concentration of PCT-65 in the hippocampus by Northernblot analysis, we decided to screen a rat hippocampal cDNA library usingthe PCT-65₁ fragment in order to obtain a full-length clone. Libraryscreening methods are well known in the art and preferred screeningmethods are disclosed in Example 3. One positive clone having a cDNAinsert of ˜2.9 kb was isolated. The complete nucleotide sequence wasdetermined by the Sanger dideoxy nucleotide chain termination methodusing the commercially available Sequenase™ kit (US BiochemicalCorporation).

The PCT-65 clone isolated from the rat hippocampal library screen wascleaved with EcoR1 to obtain a 1.5 kb fragment, PCT-65₂, (SEQ ID NO: 5)that was subcloned into pBluescript SKII (+) (Stratagene) followingtechniques provided by Stratagene. This EcoR1 fragment contained an openreading frame having sequences homologous to transmembrane regions I-VIIof previously cloned serotonin receptors 5HT-1A, 5HT-1B, 5HT-1C, 5HT-1D,5HT-1E and 5HT2. Careful inspection of the sequence further revealed thepresence of putative intronic donor and acceptor sequences. Thissuggested the possibility that the rat hippocampal cDNA represented anincompletely-spliced RNA transcript.

The possibility that this intron was removed in the functional PCT-65receptor protein was investigated by using PCR on a fresh preparation ofrat hippocampal mRNA to amplify the putative intron region. Briefly,messenger RNA was purified from a fresh preparation of rat hippocampaltissue by standard, well known methods. The mRNA was converted intodouble stranded cDNA by reverse transcription using an oligo dT primerfollowed by second strand systhesis with DNA polymerase. PCR procedureson the cDNA were performed by the method disclosed in Example 1.Amplification across the intron region using primers P1 (SEQ ID NO: 3)and P2 (SEQ ID NO: 4) resulted in two cDNA products.

One cDNA corresponded to an unspliced RNA with a sequence similar toPCT65₂, whereas the other (PCT-65_(2a)) was an RNA molecule with theintron removed. Sequencing of the spliced cDNA (PCT-65_(2a)) confirmedour hypothesis that the putative intron was removed in the active PCT-65receptor protein. The sequence of primer SEQ ID NO: 4 (below) iscomplementary to the coding sequence since it was designed to hybridizewith the opposite nucleotide strand.

P1 5'-GATCTTCGGC CACTTCTTCT GCAACGTCTT-3'(SEQ ID NO: 3)

P2 5'-TGGGAACTTG TGTTTGGCTG CACTCTTCCT-3'(SEQ ID NO: 4)

This result suggested that there were two forms of RNA in thehippocampus with only one of the RNA forms being completely spliced. Aproperly spliced version of the PCT-65 receptor is shown in SEQ ID NO: 6and the corresponding protein is shown in SEQ ID NO: 7.

We compared the homologous regions between PCT-65 and the Drosophila 5HTreceptor (Dro-1), 5HT_(1A), 5HT_(1B), 5HT_(1C), 5HT_(1D), 5HT_(1E),5HT₂, the 5HT-receptor like (SRL) from stomach fundus tissue, and anovel 5HT receptor from rat striatum (STB-17). Within the transmembraneregions, the PCT-65 receptor is between 37%-56% homologous to previouslycloned 5HT receptors. The homologies indicated that although the PCT-65receptor has significant sequence similarities to the previously clonedserotonin receptor subtypes, sequence differences are plainly evident.

EXPRESSION OF PCT-65 ENCODING PROTEIN IN MAMMALIAN CELLS

To provide the assays necessary for studying the pharmacological bindingpattern of drugs which affect serotonin receptors, PCT-65 was expressedin eukaryotic cells. There are a variety of expression vectors and celltypes available in the art to facilitate protein expression. In theembodiment disclosed in Example 3, PCT-65 was inserted into the pSRαmammalian expression plasmid (Takabe et al., (1988) Molec. Cell Biol. 8:466-472) and transfected into mammalian COS-7 cells.

To properly express the PCT-65 receptor, the PCT-65₂ fragment wasmodified to remove the intron sequence by the procedure outlined in FIG.2. Briefly, the PCT-65₂ fragment, containing the entire gene sequenceand intron was digested with Bcl 1 (Boehringer Mannheim, Indianapolis,Ind.). The PCT-65_(2a) fragment (having a properly spliced regionbetween the Bcl 1 sites) was digested and ligated back together with theBcl 1 fragments from PCT-65₂. The reconstructed PCT-65 receptor gene(with the full coding sequence and no intron) was subcloned into pSRα atthe EcoR1 site.

The resulting construct (pSRα-65) was transfected by calciumphosphate-mediated transfection into Cos-7 cells (American Type CultureCollection, Rockville Md.) using the methods described in Sambrook etal. (Molecular Cloning: A Laboratory Manual (1990) Cold Spring Harbor,N.Y.).

ASSAYS TO DETERMINE PCT-65 SEROTONIN RECEPTOR INHIBITORY DRUGS

The molecular cloning and expression of the PCT-65 serotonin receptorwill be useful in the development of drugs which interact at thisbinding site. For instance, the cloning of the PCT-65 receptor proteinprovided new assays for discovering serotonin receptor binding andinhibitory drugs. These assays were performed by transfecting Cos-7cells, as discussed above, and placing them in media to express thePCT-65 receptor on their cell surface (Example 4). The cells were lysedand the membranes isolated for binding studies. Radiolabeled ligandswere then tested for binding to the isolated membranes from these cells.

By using this method we were able to screen many drugs for their bindingactivity to the serotonin receptor. Similarly, this assay system wasused to detect competitive inhibitors of serotonin binding. Incubationof the PCT-65 transfected cells in the presence of ³ H!-LSD, a known5-HT binding molecule, led to our determination of LSD's binding andsaturation point for the PCT-65 receptor. These experiments arediscussed in Example 4 and illustrated in FIG. 3a-1. Approximate ³H!-LSD concentrations o9f 10 nM to 12 nM are shown to saturate thePCT-65 receptor. The standard saturation curve underscores ourcontention that PCT-65 is a serotonin receptor protein.

Since serotonin binding is intimately involved in the functionality ofthe central nervous system, assays for detecting drugs which bind orblock serotonin receptors are of paramount importance. As discussed inthe background, many behavioral functions are believed to be mediatedthrough serotonergic systems. For researchers to accurately assess theeffects of a new serotonin related drug in humans, it is important totest that drug's effect on every known serotonin receptor. The discoveryof the PCT-65 serotonin receptor provides the researcher with apreviously unknown assay to study possible in vivo effects of futurecentral nervous system drugs.

This discovery thereby leads to methods of screening drug candidates forcentral nervous system activity by testing their binding to isolatedPCT-65. We believe that drugs which bind to the PCT-65 receptor in vitrowill also have effects in vivo. In addition to directly measuring thebinding of a drug to the PCT-65 receptor, we were also able to measurethe displacement of known binding ligands to the PCT-65 receptor. Bymeasuring the binding of a ligand as discussed above, and then comparingthat binding to the level of affinity in the presence of a drugcandidate, we were able to estimate the potential of the drug todisplace the ligand (Example 5).

We also anticipate that the isolated PCT-65 receptor protein will beuseful for performing assays to determine agents which inhibit bindingto serotonin receptors. In discussing the isolated PCT-65 receptor, weinclude not only purified protein Example 9, but cell membranescontaining expressed PCT-65 isolated from the cell. For example, wetransfected Cos-7 cells with the gene encoding the PCT-65 receptorprotein in an expression plasmid. After incubation in media to allowPCT-65 receptor expression, the cells were lysed and the membranes(containing the bound PCT-65 receptors) were pelleted. These membranes,isolated from the host cells, were used for radiolabeled ligand bindingstudies (Example 5).

Assays provided by the present invention involve the utilization of avariety of drugs which are known to exhibit specificity for variousserotonergic receptor subtypes and other binding sites. The averageconstant of inhibition (K_(i)) values for compounds competing withbetter than 10 μM affinity are shown in Table 1 with representativecompetition curves for ³ H!-LSD binding shown in FIG. 3b.

Examination of the rank order of potency for a variety of serotonergicagents reveals that the pharmacology of the PCT-65 receptor does notcorrespond to any previously described serotonin receptor subtype. Anumber of drugs selective for other 5-HT receptor subtypes such as8-OH-DPAT (5HT_(1A)) bind with relatively low affinity. Ergot alkaloids,especially ergoline derivatives (i.e., LSD, lisuride, or pergolide) andmesulergine (5-HT_(1C)) display relatively high affinity for PCT-65 asdoes the non-selective serotonergic antagonist methiothepin.

Interestingly, the typical anti-psychotic loxapine exhibited lowaffinity for PCT-65, while the atypical psychotic drug clozapine hadhigh affinity for PCT-65. One tricyclic anti-depressant drug(amitriptyline) had a K_(i) value under 100 nM. In general, the drugswhich exhibited the greatest affinity for PCT-65 (i.e., K_(i) <100 nM)were tricyclic, ergoline or tryptamine derivatives.

Competition for ³ H!-5-HT (serotonin) binding by a number of drugsrevealed, with a few exceptions, the same rank order of potency as forinhibition of ³ H!-LSD binding (Table 1). However, the K_(i) values forsome drugs were determined by competition with ³ H!-5-HT and found to beup to 5-fold lower than those determined by competition with ³ H!-LSD,with the exception of metergoline and clozapine which exhibited somewhatgreater potency in competition with ³ H!-LSD.

These competition assays provide methods of screening potential centralnervous system drugs for their potential pharmacological effects invivo. It is also anticipated that the PCT-65 receptor screening assayswill be very important for determining the potential dosages of putativedrugs prior to introduction in vivo.

Other assay methods are also anticipated by the present invention. Forexample, the expression of PCT-65 on the cell surface can be detected byeither polyclonal or monoclonal antibodies directed against PCT-65epitopes. Possible disease states wherein a defective PCT-65 receptor isexpressed could thus be detected by comparison of antibody binding ofnormal cells to binding of the disease state cells. Methods of producingantibodies against PCT-65 are discussed in Examples 7 and 8.

Additionally, the PCT-65 serotonin receptor can be purified by antibodyaffinity chromatography (Harlow et al. Antibodies--A Laboratory Manual,Cold Spring Harbor (1988) N.Y.). In one embodiment of this method,monoclonal antibodies produced in Example 9 are attached to an affinitycolumn, and contacted with transfected Cos-7 cell lysates. The boundproteins are eluted off in a low pH solution by standard methods.

In situ hybridization methods Example 9 can be used to determine mRNAexpression in rat hippocampal tissue. Briefly, rat brains are isolatedand incubated in a hybridization solution comprising radiolabeled primerP1. The labeled primer enters the sectioned brain sample and hybridizeswith the cytoplasmic mRNA resulting in a detectable signal on anautoradiograph. It is also contemplated that other methods of labelingthe PCT-65 specific oligonucleotide are within the scope of thisinvention. Other methods of labeling could be colorimetric or enzymatic.

Particular embodiments of the invention will be discussed in detail andreference will be made to possible variations within the scope of theinvention. There are a variety of alternative techniques and proceduresavailable to those of skill in the art which would similarly permit oneto obtain the elements of this invention.

EXAMPLE 1 Identification of the PCT-65 Clone

Total RNA was prepared from rat kidney proximal convoluted tubule (PCT)using RNazol B (CINNA/BIOTECK, Laboratories International) and poly A+RNA was isolated using poly-A Quick columns (Stratagene). First strandcDNA synthesized from the rat PCT mRNA was amplified by PCR with 1 μMeach of the following primers:

5' GTCGACCCT(GT)T(GT) (CG)GCC(AC)TCA(GT)CA(TC) (GA)G(AG)TCGCTA-3'SEQ IDNO: 1

5' -AAGCTTATGAA(AG)AAGGGCAG(GC)CA(AG)CAGAGG(TG) (CT) (AG)(CA)A-3'SEQ IDNO: 2

The PCR reaction proceeded for 1.5 min at 93° C., 2 min at 50° C., and 4min at 72° C. followed by a 7 min extension at 72° C. The reactionproducts were purified by ultrafiltration with Centricon 30 units(Amicon), digested with Sal I and Hind III, and gel isolated on a 1%agarose gel. Individual bands were excised, electroeluted, concentratedby ultrafiltration, and ligated into Sal/Hind III digested pBluescriptII SK (+) (Stratagene).

Competent DH5α cells (Invitrogen) were transformed and minipreparationsof the plasmid DNA was prepared for insert sequencing using the Sangerdideoxy nucleotide chain termination method with Sequenase (USBiochemical Corp.) One clone PCT-65₁ having homology to other serotoninreceptors was chosen for further study.

EXAMPLE 2 Tissue Distribution of PCT-65 by Northern Blot Analysis

Poly (A)⁺ RNA was isolated from rat brain and peripheral tissues.Samples (1 μg in each lane) were denatured and electrophoresed through1% agarose gel having 0.66 M formaldehyde, then transferred to nylonmembrane (GeneScreen Plus, DuPont) by capillary transfer and immobilizedby UV cross-linking. Northern blots were probed with PCT-65₁ PCR productamplified from the region between TM III and TM VI and ³² P-labelled bythe random primer method. The hybridization was carried out using 1×10⁶dpm/ml of the probe in 1 M NaCl, 1% SDS, 50% formamide, 10% DextranSulfate at 60° C. for 18 hr. Blots were washed in 2X SSC at roomtemperature, followed by 2X SSC with 1% SDS at 65° C., and then exposedfor 15 days at -70° C. to X-ray film with an intensifying screen.

EXAMPLE 3 Identification of a Full Length PCT-65 Clone

1×10⁶ recombinants from a rat hippocampal cDNA library, constructed inthe λ gt 11 vector (Stratagene), were screened with a PCR fragmentPCT-65₁ which was ³² P-labeled via nick translation. Duplicatenitrocellulose filters were hybridized in 50% formamide, 0.75 MNaCl/0.075 M sodium citrate (5X SSC), 5X Denhardt's solution, 0.1% Na₂HPO₄, 01.% SDS, 0.15 mg/ml salmon sperm DNA, and 1.5×10⁸ dpm/ml of ³²P-labeled probe for 36 hr at 42° C.

The filters were washed at high stringency conditions: 2X SSC and 0.1%SDS at room temperature for 30 min and with 0.2X SSC and 1% SDS at 68°C. for 1 hour prior autoradiography. λ phage found to hybridize to theprobe were subsequently plaque purified. One positive clone contained a2.9 kb insert, from which a 1.5 kb fragment was removed with EcoR1 andsubcloned into pBluescript SK II(+) by standard methods. The 1.5 kbEcoR1 fragment (PCT-65₂ ; SEQ ID NO: 5) was nucleotide sequenced usingthe Sanger dideoxy method.

As discussed above, a 73 nucleotide intron was discovered beginning atnucleotide +415 of PCT-65₂. Example 4 below shows the method we used toanalyze the expression and binding properties of PCT-65.

EXAMPLE 4 Ligand Binding Assays Using the PCT-65 Receptor

We removed the intron of clone PCT-65₂ by digesting with Bcl 1. Thiscloning procedure is illustrated in FIG. 2. Briefly, we digested thespliced version (ie: without intron) of the PCT-65 cDNA (PCT-65_(2a))with Bcl 1 and mixed these fragments and those derived from the Bcl 1digestion of PCT-65₂. The fragment pool was incubated and ligated bywell known methods. Fragments having EcoR1 sites were ligated intopBluescript and nucleotide sequenced.

One clone (PCT-65 ) having the entire coding sequence, but missing theintron, was subcloned into the expression vector pSRα. This plasmidconstruct (pSRα-65) was introduced into COS-7 cells by calciumphosphate-mediated transfection. Cells were harvested 3 days aftertransfection in 5 mM Tris-HCl, pH 7.4 at 37° C., 5 mM MgCl₂, 250 mMsucrose, and stored in liquid N₂ prior to membrane preparation.

Crude membranes were prepared from cell homogenates by centrifugation at43,000×g for 10 min and the resulting pellets resuspended in the bindingbuffer: 50 mM Tris-HCl, 1.5 mM CaCl₂, 4.0 mM MgCl₂, 5.0 mM KCl, 120 mMNaCl and 1.0 mM EDTA, pH7.4, 25° C., at a protein concentration of 25-35μg/ml.

For saturation experiments (FIG. 3a), 0.1 ml of the membrane suspensionwas incubated in a final volume of 1 ml with increasing concentrationsof ³ H!-LSD (67 Ci/mMole, 78-10,000 pM), 200 μM sodium metabisulfate intriplicate for 60 min at 37° C. 100 μM of 5-HT was utilized to determinenon-specific binding. FIG. 3a-1 illustrates the saturation binding of ³H!-LSD to the PCT-65 receptor.

EXAMPLE 5 Pharmacological Competition Studies Using the PCT-65 SerotoninReceptor

Table 1 (below) shows varying concentrations of of the indicatedcompeting ligands (10⁻¹¹ M-10⁻⁵ M) which were utilized to study thebinding inhibition of ³ H!-LSD (1.5-2.2 nM) or ³ H!-5-HT (1 nM) tomembranes prepared from the COS-7 cells transiently transfected withclone PCT-65 as explained in Example 4.

Inhibition constant (Ki) values (Table 1) were obtained by correction ofgraphically determined IC₅₀ values according the method of Cheng andPrussof (Biochem. Pharmacol. 22, 3099-3108 (1973)) and are presented asthe geometric mean±SEM (n=3)

Competition assays were initiated by addition of 0.1 ml of the membranesuspension and incubated in triplicate with about 2 nM ³ H!-LSD or about1 nM ³ H!-5-HT, 200 μM sodium metabisulfite, and increasingconcentrations of competing ligands (10⁻¹¹ to 10³¹ 5 M) at 37° C. for 60min.

The assays were terminated by rapid filtration through Whatman GF/Cfilters pretreated with 0.3% polyethy-leneimine and washed four timeswith 3 ml of 50 mM Tris-HC1, pH7.4, at 4° C. Radioactivity levels weredetermined by liquid scintillation counting at an efficiency of 47%.Competition experiments for the ³ H!-5HT were performed in triplicate asdescribed by Hamblin et al. (Molec. Pharm. (1991) 40: 143-148).

Specifically, transfected cells were scraped into PBS with 5 mM EDTA,spun, polytroned, spun, suspended in 100 mM Tris, 20 mM MgSO4, 1 mMEDTA, pH7.7. 0.5 ml of this solution containing 10-25 μg protein wasadded to 0.5 ml of a 2 mM ascorbic acid solution containing the ligandand drugs and incubated for 30 min at 37° C. Filtration was performed onGF/C paper (0.1% PEI solution pre-treatment) with a 10 ml rinse.

The following compounds which are not in Table 1 were tested and foundto have Ki values >1.3 mM: Imipramine, NAN-190, Desipramine, Citalopram,Fluoxetine, Idazoxan, Quipazine, LY-278584, Octopamine, MDL7222,BRL24294, BRL43694, BIMU1, BIMU8, DAU6215, DAU6285, GR38032, Zacopride,Fenflurmine, Pindolol, Dopamine, Norepinephrine, Histamine, andMelotonin.

                  TABLE 1    ______________________________________    5HT and  3-H!  for  3H!    binding to the PCT-65 receptor expressed in COS-7 cells                   Ki (nM) ± SEM    LSDgs            vs.  .sup.3 H!    5-HT                        vs.  .sup.3 H!    ______________________________________    5-Carboxamidotryptamine                      0.33 ± 0.01                                 0.16 ± 0.05    Lisuride          0.52 ± 0.12                                 0.89 ± 0.32    Methiothepin      1.03 ± 0.16                                 0.38 ± 0.18    5-Methoxytryptamine                      1.78 ± 0.05                                 0.57 ± 0.03    5-Hydroxytryptamine                      1.83 ± 0.07                                 1.52 ± 0.27    Metergoline       2.04 ± 0.15                                 6.21 ± 1.00    Pergolide         4.78 ± 0.18                                 1.01 ± 0.03    Mesulergine       7.13 ± 0.39                                 21.1 ± 2.19    Bromocriptine    10.72 ± 0.47                                48.80 ± 7.90    Methysergide     12.55 ± 0.84                                13.40 ± 0.80    Clozapine        13.55 ± 2.61                                39.50 ± 9.60    5-Methoxy-N,     20.93 ± 0.86                                 8.26 ± 0.73    N-dimethytryptamine    Ritanserin       21.71 ± 2.10                                15.00 ± 3.40    Tryptamine       32.85 ± 0.76                                16.42 ± 1.01    1(1-Napthyl)piperazine                     33.75 ± 1.68                                18.39 ± 0.77    8-OH-DPAT        35.44 ± 5.24                                35.00 ± 5.62    Cyproheptidine   48.01 ± 3.26                                34.10 ± 4.15    Mianserin        111.60 ± 13.84                                36.90 ± 2.60    Amitriptyline    123.97 ± 13.37                                 92.80 ± 15.80    Loxapine         171.53 ± 7.80                                133.00 ± 36.10    Ketanserin       206.79 ± 19.56                                265.00 ± 32.10    2-MPP            243.59 ± 16.47                                125.00 ± 8.40    mCPP             352.05 ± 54.91                                256.00 ± 9.90    TFMPP            532.93 ± 33.04                                236.00 ± 5.50    Sumatriptan                 251.00 ± 36.10    ______________________________________

Since Cos-7 cells are only transiently transfected, we stablytransfected Human Embryonic Kidney cells (HEK 293 available from theATCC) in 150 mm dishes by the CaPO₄ technique with 30 μg of PCT-65 (SEQID NO: 6) and 3 μg of pMam-neo (INVITROGEN). These HEK 293 cells werethen subjected to selection with 600 μg/ml genetecin (G418) (GIBCO). Theresistant colonies were collected and screened for receptor expressionby ³ H!-LSD binding as described in Example 4. Assays using transfectedHEK-293 cells revealed similar saturation and drug inhibitory patternsas that found with transfected Cos-7 cells.

We were also interested in isolating the human variation of the PCT-65gene sequence. Our method of isolating the human receptor can be foundin the following example.

EXAMPLE 6 Isolation of the Human Receptor

A 1349 bp NotI-EcoRI restriction fragment derived from the rat 5-HT₇receptor cDNA pct65 was labeled with α³² P!-dCTP using thenick-translation method and used to screen a commercially availablehuman hypothalamic cDNA library (Clontech #29068) constructed in theviral vector λGT10. The library was plated using the host cell C600,followed by duplicate nylon (Nytran, Schleicher and Schuell) lifts.

Probe hybridization (6 X SSC at 55° C., pH7) and washing (2 X SSC, 58°C.) were then performed by well known methods at moderate stringency.Positive isolates were plaque purified through a second round ofhybridization. In the second round, cDNA inserts from purified isolateswere excised with EcoRI and then subcloned into the corresponding sitein the phagemid vector pBluescript SK(-) (Stratagene). The nucleic acidsequence of these inserts were then determined by dideoxynucleotidetermination using Sequenase (United States Biochemicals) in conjunctionwith standard subcloning techniques. This method allowed the sequencingof vitually the entire gene. Only about 117 nucleotides of the 5' endare missing. The nucloetide sequence of the majority of human PCT-65receptor is SEQUENCE ID NO: 8 which encodes the protein of SEQUENCE IDNO: 9. One of ordinary skill in the art could use these same methods todetermine the remaining nucleotide sequences of the 5' end.

It should be recognized that all of the previous experiments relating toidentification of binding ligands to the rat receptor can be performedin a similar fashion with the human gene. For instance, the human genecan be cloned into an expression plasmid and placed in COS-7 cells bymethods similar to those discussed above, and used to assay binding ofcompetitors such as those revealed in Table 1. This would provide amethod of assaying inhibitors of serotonin binding to the PCT-65receptor.

Other experiments are also contemplated relating to discovering whichcells in vivo express the receptor gene on their surface. One method ofstudying the expression of the PCT-65 receptor on cell surfacesthroughout the body, is to produce antibodies against the receptorprotein. A method for producing polyclonal antibodies is discussed belowin Example 7.

EXAMPLE 7 Production of Antibodies Against PCT-65

COS-7 cells expressing the PCT-65 receptor protein derived in Example 4are lysed with NP40, and the isolated membranes are injected intorabbits. The lysed membranes are isolated in a non-ionic detergent so asnot to affect the membrane bound receptors. Freunds adjuvant is used inthe injection to help stimulate an antigenic response by the rabbits.After two booster shots of the lysed membranes, the rabbits are bled andthe sera isolated by centrifugation.

The antibodies in the crude rabbit sera extract are ¹²⁵ I labeled bywell known methods, and tested for activity against the transfectedCOS-7 cells. A western blot having one lane containing proteins fromtransfected cell lysates, and a second lane having untransfected lysates(control) is run. A strong band indicating antibody binding in thetransfected cell lane, that is not apparent in the untransfected lanedemonstrates that polyclonal antibodies against the PCT-65 receptorprotein have been properly isolated.

Monoclonal antibodies can be produced by well known methods in additionto the polyclonal antibodies discussed above. One method of producingmonoclonal antibodies is discussed below in Example 8.

EXAMPLE 8 Production of Monoclonal Antibodies Against PCT-65

The PCT-65 transfected COS-7 cells produced in the previous examples arelysed with NP-40 and the cell membranes are pelleted by centrifugation.The isolated membranes, having bound PCT-65 receptor proteins, areco-injected with Freunds adjuvant into mice. After being re-injected 9times over a three week period, the murine spleens are removed andresuspended in PBS.

The suspended spleen cells are mixed (approximately 4:1) with SP 2/0Myeloma cells. Polyethylene glycol is added to fuse the myeloma cells tothe spleen cells, and the fused cells are selected in HAT media. Thefused cells are aliquoted so that only one cell is grown in each well ofa 96 well microtiter plate. Each cell is grown, the media removed, andsecreted proteins are ¹²⁵ I labeled. The labeled media from each well isused to probe a Western blot of transfected and untransfected COS-7 celllysates (see Example 4).

The desired fusion cell produces a monoclonal antibody that stronglybinds a band in the transfected COS-7 cell lane on the Western blot, butdoesn't bind to any other protein in that lane, or the control lane.This method thereby discloses a procedure for detecting those cellsexpressing the PCT-65 serotonin receptor.

Another method of detecting the expression of PCT-65 is through in situhybridization as discussed below in Example 9.

EXAMPLE 9 In situ Hybridization of PCT-65

In situ hybridization allows the identification of mRNA within intacttissues, such as the rat hippocampus. In this method, oligonucleotidescorresponding to unique portions of the PCT-65 gene (SEQ ID NO: 6) areused to detect specific mRNA species in the hippocampus.

An anesthetized rat is transcardially perfused with cold PBS (5-20minutes), followed by perfusion with a 4% formaldehyde solution. Thebrain is removed, frozen in liquid nitrogen, and cut into 5 μm to 30 μmsections. The sections are placed on slides and incubated in proteinaseK for approximately 15 minutes. The slides are then rinsed in DEP,water, ethanol, and placed in a prehybridization buffer.

A radioactive probe corresponding to primer P1 is produced by nicktranslation and incubated with the sectioned hippocampal tissue. Afterincubation and air drying the labeled areas are visualized byautoradiography. Dark spots on the autoradiograph corresponding to thetissue sample indicate hybridization of the probe with the hippocampalmRNA thereby demonstrating expression of the PCT-65 receptor.

While particular embodiments of the invention have been described indetail, it will be apparent to those skilled in the art that theseembodiments are exemplary, rather than limiting, and the true scope ofthe invention is that defined in the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 9    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 33 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #         33       MTCA KCAYRGRTCG CTA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 35 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: YES    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #       35         GCAG SCARCAGAGG KYRMA    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #           30     TTCT GCAACGTCTT    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: YES    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #           30     GCTG CACTCTTCCT    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1505 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -    (vii) IMMEDIATE SOURCE:    #WITH INTRON) CLONE: PCT-65 cDNA    -     (ix) FEATURE:              (A) NAME/KEY: exon              (B) LOCATION: 126..539    -     (ix) FEATURE:              (A) NAME/KEY: intron              (B) LOCATION: 540..611    -     (ix) FEATURE:              (A) NAME/KEY: exon              (B) LOCATION: 612..1409    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - CCTCCCCAGC GGCCGCCCCG ACCTCTCTAC GGCCATCTCC GTTCACTCAT CC - #TGCCGGAG      60    - TGGGGCATGC TGGGCTGCAG GACCTGAGCC CGACGGTGGC ATGCCCACCT GT - #GGTGAGCT     120    - CCTGGATGCC GCACCTGCTG AGTGGCTTCC TAGAGGTGAC GGCTAGCCCG GC - #GCCCACCT     180    - GGGACGCGCC ACCGGACAAT GTCTCAGGCT GCGGGGAGCA GATCAACTAT GG - #CAGAGTGG     240    - AGAAAGTTGT GATCGGCTCC ATCCTGACGC TCATCACGCT GCTGACGATC GC - #AGGCAACT     300    - GCCTGGTGGT GATCTCGGTG TCCTTCGTCA AGAAGCTCCG CCAGCCCTCC AA - #CTACCTGA     360    - TTGTGTCCCT GGCCCTGGCT GACCTCTCGG TGGCCGTGGC GGTCATGCCT TT - #CGTTAGTG     420    - TCACCGACCT CATCGGGGGC AAGTGGATCT TCGGCCACTT CTTCTGCAAC GT - #CTTCATCG     480    - CCATGGACGT CATGTGCTGC ACGGCCTCGA TCATGACCCT GTGCGTGATC AG - #CATCGACA     540    - GTCTGGCTTC GGTGTTTGAC CATGCTGTCG GTCTCCTGCT TACTGACTGG AG - #AGCCTTAC     600    - TCTCGTCTTA AAGGTACCTT GGGATCACGA GACCCCTCAC GTACCCGGTG AG - #GCAAAATG     660    - GGAAATGTAT GGCCAAAATG ATTCTGTCGG TCTGGCTGCT CTCTGCCTCC AT - #CACCTTAC     720    - CTCCTCTCTT CGGATGGGCT CAGAATGTGA ACGATGACAA AGTGTGCTTG AT - #CAGCCAGG     780    - ATTTTGGCTA CACGATCTAC TCCACTGCGG TGGCGTTTTA TATCCCCATG TC - #GGTCATGC     840    - TGTTCATGTA CTATCAGATT TACAAGGCCG CCAGGAAGAG TGCAGCCAAA CA - #CAAGTTCC     900    - CAGGCTTCCC ACGCGTGCAG CCGGAGAGTG TCATCTCCCT GAATGGTGTG GT - #GAAGCTCC     960    - AGAAGGAGGT GGAAGAGTGT GCGAACCTTT CGAGACTGCT CAAACACGAA AG - #GAAAAACA    1020    - TCTCCATCTT CAAGCGGGAA CAGAAAGCAG CCACTACCTT GGGGATCATC GT - #GGGAGCCT    1080    - TCACTGTGTG CTGGCTGCCG TTTTTCCTCT TGTCCACAGC CCGCCCCTTT AT - #CTGTGGCA    1140    - CCTCCTGTAG CTGCATTCCT CTGTGGGTGG AGAGGACATG TCTGTGGCTG GG - #CTATGCAA    1200    - ACTCTCTCAT TAATCCTTTT ATATATGCCT TCTTCAACCG GGACCTGAGG CC - #CACCTCTC    1260    - GTAGCCTACT CCAGTGCCAG TACCGGAATA TCAACCGGAA GCTCTCTGCT GC - #AGGCATGC    1320    - ATGAAGCCCT GAAACTTGCT GAGAGGCCCG AGAGATCCGA GTTTGTGCTA CA - #AAACTCTG    1380    - ACCACTGTGG GAAAAAGGGT CATGATACAT GATCCAGAGT GGAACCCTGG AT - #GAATTCAT    1440    - GCAGAACAGG TGGACACAAC ACAACGAATC ATTGGCTGAG ACTGCACATG GA - #CATGCTCT    1500    #          1505    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1433 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 126..1337    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - CCTCCCCAGC GGCCGCCCCG ACCTCTCTAC GGCCATCTCC GTTCACTCAT CC - #TGCCGGAG      60    - TGGGGCATGC TGGGCTGCAG GACCTGAGCC CGACGGTGGC ATGCCCACCT GT - #GGTGAGCT     120    #GAG GTG ACG GCT AGC        167 GGC TTC CTA    #Phe Leu Glu Val Thr Ala Serr Gly    #     10    - CCG GCG CCC ACC TGG GAC GCG CCA CCG GAC AA - #T GTC TCA GGC TGC GGG     215    Pro Ala Pro Thr Trp Asp Ala Pro Pro Asp As - #n Val Ser Gly Cys Gly    # 30    - GAG CAG ATC AAC TAT GGC AGA GTG GAG AAA GT - #T GTG ATC GGC TCC ATC     263    Glu Gln Ile Asn Tyr Gly Arg Val Glu Lys Va - #l Val Ile Gly Ser Ile    #                 45    - CTG ACG CTC ATC ACG CTG CTG ACG ATC GCA GG - #C AAC TGC CTG GTG GTG     311    Leu Thr Leu Ile Thr Leu Leu Thr Ile Ala Gl - #y Asn Cys Leu Val Val    #             60    - ATC TCG GTG TCC TTC GTC AAG AAG CTC CGC CA - #G CCC TCC AAC TAC CTG     359    Ile Ser Val Ser Phe Val Lys Lys Leu Arg Gl - #n Pro Ser Asn Tyr Leu    #         75    - ATT GTG TCC CTG GCC CTG GCT GAC CTC TCG GT - #G GCC GTG GCG GTC ATG     407    Ile Val Ser Leu Ala Leu Ala Asp Leu Ser Va - #l Ala Val Ala Val Met    #     90    - CCT TTC GTT AGT GTC ACC GAC CTC ATC GGG GG - #C AAG TGG ATC TTC GGC     455    Pro Phe Val Ser Val Thr Asp Leu Ile Gly Gl - #y Lys Trp Ile Phe Gly    #110    - CAC TTC TTC TGC AAC GTC TTC ATC GCC ATG GA - #C GTC ATG TGC TGC ACG     503    His Phe Phe Cys Asn Val Phe Ile Ala Met As - #p Val Met Cys Cys Thr    #               125    - GCC TCG ATC ATG ACC CTG TGC GTG ATC AGC AT - #C GAC AGG TAC CTT GGG     551    Ala Ser Ile Met Thr Leu Cys Val Ile Ser Il - #e Asp Arg Tyr Leu Gly    #           140    - ATC ACG AGA CCC CTC ACG TAC CCG GTG AGG CA - #A AAT GGG AAA TGT ATG     599    Ile Thr Arg Pro Leu Thr Tyr Pro Val Arg Gl - #n Asn Gly Lys Cys Met    #       155    - GCC AAA ATG ATT CTG TCG GTC TGG CTG CTC TC - #T GCC TCC ATC ACC TTA     647    Ala Lys Met Ile Leu Ser Val Trp Leu Leu Se - #r Ala Ser Ile Thr Leu    #   170    - CCT CCT CTC TTC GGA TGG GCT CAG AAT GTG AA - #C GAT GAC AAA GTG TGC     695    Pro Pro Leu Phe Gly Trp Ala Gln Asn Val As - #n Asp Asp Lys Val Cys    175                 1 - #80                 1 - #85                 1 -    #90    - TTG ATC AGC CAG GAT TTT GGC TAC ACG ATC TA - #C TCC ACT GCG GTG GCG     743    Leu Ile Ser Gln Asp Phe Gly Tyr Thr Ile Ty - #r Ser Thr Ala Val Ala    #               205    - TTT TAT ATC CCC ATG TCG GTC ATG CTG TTC AT - #G TAC TAT CAG ATT TAC     791    Phe Tyr Ile Pro Met Ser Val Met Leu Phe Me - #t Tyr Tyr Gln Ile Tyr    #           220    - AAG GCC GCC AGG AAG AGT GCA GCC AAA CAC AA - #G TTC CCA GGC TTC CCA     839    Lys Ala Ala Arg Lys Ser Ala Ala Lys His Ly - #s Phe Pro Gly Phe Pro    #       235    - CGC GTG CAG CCG GAG AGT GTC ATC TCC CTG AA - #T GGT GTG GTG AAG CTC     887    Arg Val Gln Pro Glu Ser Val Ile Ser Leu As - #n Gly Val Val Lys Leu    #   250    - CAG AAG GAG GTG GAA GAG TGT GCG AAC CTT TC - #G AGA CTG CTC AAA CAC     935    Gln Lys Glu Val Glu Glu Cys Ala Asn Leu Se - #r Arg Leu Leu Lys His    255                 2 - #60                 2 - #65                 2 -    #70    - GAA AGG AAA AAC ATC TCC ATC TTC AAG CGG GA - #A CAG AAA GCA GCC ACT     983    Glu Arg Lys Asn Ile Ser Ile Phe Lys Arg Gl - #u Gln Lys Ala Ala Thr    #               285    - ACC TTG GGG ATC ATC GTG GGA GCC TTC ACT GT - #G TGC TGG CTG CCG TTT    1031    Thr Leu Gly Ile Ile Val Gly Ala Phe Thr Va - #l Cys Trp Leu Pro Phe    #           300    - TTC CTC TTG TCC ACA GCC CGC CCC TTT ATC TG - #T GGC ACC TCC TGT AGC    1079    Phe Leu Leu Ser Thr Ala Arg Pro Phe Ile Cy - #s Gly Thr Ser Cys Ser    #       315    - TGC ATT CCT CTG TGG GTG GAG AGG ACA TGT CT - #G TGG CTG GGC TAT GCA    1127    Cys Ile Pro Leu Trp Val Glu Arg Thr Cys Le - #u Trp Leu Gly Tyr Ala    #   330    - AAC TCT CTC ATT AAT CCT TTT ATA TAT GCC TT - #C TTC AAC CGG GAC CTG    1175    Asn Ser Leu Ile Asn Pro Phe Ile Tyr Ala Ph - #e Phe Asn Arg Asp Leu    335                 3 - #40                 3 - #45                 3 -    #50    - AGG CCC ACC TCT CGT AGC CTA CTC CAG TGC CA - #G TAC CGG AAT ATC AAC    1223    Arg Pro Thr Ser Arg Ser Leu Leu Gln Cys Gl - #n Tyr Arg Asn Ile Asn    #               365    - CGG AAG CTC TCT GCT GCA GGC ATG CAT GAA GC - #C CTG AAA CTT GCT GAG    1271    Arg Lys Leu Ser Ala Ala Gly Met His Glu Al - #a Leu Lys Leu Ala Glu    #           380    - AGG CCC GAG AGA TCC GAG TTT GTG CTA CAA AA - #C TCT GAC CAC TGT GGG    1319    Arg Pro Glu Arg Ser Glu Phe Val Leu Gln As - #n Ser Asp His Cys Gly    #       395    - AAA AAG GGT CAT GAT ACA TGATCCAGAG TGGAACCCTG GA - #TGAATTCA    1367    Lys Lys Gly His Asp Thr        400    - TGCAGAACAG GTGGACACAA CACAACGAAT CATTGGCTGA GACTGCACAT GG - #ACATGCTC    1427    #         1433    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 404 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - Met Pro His Leu Leu Ser Gly Phe Leu Glu Va - #l Thr Ala Ser Pro Ala    #                 15    - Pro Thr Trp Asp Ala Pro Pro Asp Asn Val Se - #r Gly Cys Gly Glu Gln    #             30    - Ile Asn Tyr Gly Arg Val Glu Lys Val Val Il - #e Gly Ser Ile Leu Thr    #         45    - Leu Ile Thr Leu Leu Thr Ile Ala Gly Asn Cy - #s Leu Val Val Ile Ser    #     60    - Val Ser Phe Val Lys Lys Leu Arg Gln Pro Se - #r Asn Tyr Leu Ile Val    # 80    - Ser Leu Ala Leu Ala Asp Leu Ser Val Ala Va - #l Ala Val Met Pro Phe    #                 95    - Val Ser Val Thr Asp Leu Ile Gly Gly Lys Tr - #p Ile Phe Gly His Phe    #           110    - Phe Cys Asn Val Phe Ile Ala Met Asp Val Me - #t Cys Cys Thr Ala Ser    #       125    - Ile Met Thr Leu Cys Val Ile Ser Ile Asp Ar - #g Tyr Leu Gly Ile Thr    #   140    - Arg Pro Leu Thr Tyr Pro Val Arg Gln Asn Gl - #y Lys Cys Met Ala Lys    145                 1 - #50                 1 - #55                 1 -    #60    - Met Ile Leu Ser Val Trp Leu Leu Ser Ala Se - #r Ile Thr Leu Pro Pro    #               175    - Leu Phe Gly Trp Ala Gln Asn Val Asn Asp As - #p Lys Val Cys Leu Ile    #           190    - Ser Gln Asp Phe Gly Tyr Thr Ile Tyr Ser Th - #r Ala Val Ala Phe Tyr    #       205    - Ile Pro Met Ser Val Met Leu Phe Met Tyr Ty - #r Gln Ile Tyr Lys Ala    #   220    - Ala Arg Lys Ser Ala Ala Lys His Lys Phe Pr - #o Gly Phe Pro Arg Val    225                 2 - #30                 2 - #35                 2 -    #40    - Gln Pro Glu Ser Val Ile Ser Leu Asn Gly Va - #l Val Lys Leu Gln Lys    #               255    - Glu Val Glu Glu Cys Ala Asn Leu Ser Arg Le - #u Leu Lys His Glu Arg    #           270    - Lys Asn Ile Ser Ile Phe Lys Arg Glu Gln Ly - #s Ala Ala Thr Thr Leu    #       285    - Gly Ile Ile Val Gly Ala Phe Thr Val Cys Tr - #p Leu Pro Phe Phe Leu    #   300    - Leu Ser Thr Ala Arg Pro Phe Ile Cys Gly Th - #r Ser Cys Ser Cys Ile    305                 3 - #10                 3 - #15                 3 -    #20    - Pro Leu Trp Val Glu Arg Thr Cys Leu Trp Le - #u Gly Tyr Ala Asn Ser    #               335    - Leu Ile Asn Pro Phe Ile Tyr Ala Phe Phe As - #n Arg Asp Leu Arg Pro    #           350    - Thr Ser Arg Ser Leu Leu Gln Cys Gln Tyr Ar - #g Asn Ile Asn Arg Lys    #       365    - Leu Ser Ala Ala Gly Met His Glu Ala Leu Ly - #s Leu Ala Glu Arg Pro    #   380    - Glu Arg Ser Glu Phe Val Leu Gln Asn Ser As - #p His Cys Gly Lys Lys    385                 3 - #90                 3 - #95                 4 -    #00    - Gly His Asp Thr    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1417 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -    (vii) IMMEDIATE SOURCE:              (B) CLONE: Human PCT-65    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 8..1105    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #CTG ACG CTC ATC ACG       49 ATC GGC TCC ATC    #Gly Ser Ile Leu Thr Leu Ile Thr    #       10    - CTG CTG ACG ATC GCG GGC AAC TGC CTG GTG GT - #G ATC TCC GTG TGC TTC      97    Leu Leu Thr Ile Ala Gly Asn Cys Leu Val Va - #l Ile Ser Val Cys Phe    # 30    - GTC AAG AAG CTC CGC CAG CCC TCC AAC TAC CT - #G ATC GTG TCC CTG GCG     145    Val Lys Lys Leu Arg Gln Pro Ser Asn Tyr Le - #u Ile Val Ser Leu Ala    #                 45    - CTG GCC GAC CTC TCG GTG GCT GTG GCG GTC AT - #G CCC TTC GTC AGC GTC     193    Leu Ala Asp Leu Ser Val Ala Val Ala Val Me - #t Pro Phe Val Ser Val    #             60    - ACC GAC CTC ATC GGG GGC AAG TGG ATC TTT GG - #A CAC TTT TTC TGT AAT     241    Thr Asp Leu Ile Gly Gly Lys Trp Ile Phe Gl - #y His Phe Phe Cys Asn    #         75    - GTC TTC ATC GCC ATG GAC GTC ATG TGC TGC AC - #G GCC TCG ATC ATG ACC     289    Val Phe Ile Ala Met Asp Val Met Cys Cys Th - #r Ala Ser Ile Met Thr    #     90    - CTG TGC GTG ATC AGC ATT GAC AGG TAC CTT GG - #G ATC ACA AGG CCC CTC     337    Leu Cys Val Ile Ser Ile Asp Arg Tyr Leu Gl - #y Ile Thr Arg Pro Leu    #110    - ACA TAC CCT GTG AGG CAG AAT GGG AAA TGC AT - #G GCG AAG ATG ATT CTC     385    Thr Tyr Pro Val Arg Gln Asn Gly Lys Cys Me - #t Ala Lys Met Ile Leu    #               125    - TCC GTC TGG CTT CTC TCC GCC TCC ATC ACC TT - #A CCT CCA CTC TTT GGA     433    Ser Val Trp Leu Leu Ser Ala Ser Ile Thr Le - #u Pro Pro Leu Phe Gly    #           140    - TGG GCT CAG AAT GTA AAT GAT GAT AAG GTG TG - #C TTG ATC AGC CAG GAC     481    Trp Ala Gln Asn Val Asn Asp Asp Lys Val Cy - #s Leu Ile Ser Gln Asp    #       155    - TTT GGC TAT ACG ATT TAC TCT ACC GCA GTG GC - #A TTT TAT ATC CCC ATG     529    Phe Gly Tyr Thr Ile Tyr Ser Thr Ala Val Al - #a Phe Tyr Ile Pro Met    #   170    - TCC GTC ATG CTT TTC ATG TAC TAC CAG ATT TA - #C AAG GCT GCC AGG AAG     577    Ser Val Met Leu Phe Met Tyr Tyr Gln Ile Ty - #r Lys Ala Ala Arg Lys    175                 1 - #80                 1 - #85                 1 -    #90    - AGT GCT GCC AAA CAC AAG TTT CCT GGC TTC CC - #T CGA GTG GAG CCA GAC     625    Ser Ala Ala Lys His Lys Phe Pro Gly Phe Pr - #o Arg Val Glu Pro Asp    #               205    - AGC GTC ATC GCC CTG AAT GGC ATA GTG AAG CT - #C CAG AAG GAG GTG GAA     673    Ser Val Ile Ala Leu Asn Gly Ile Val Lys Le - #u Gln Lys Glu Val Glu    #           220    - GAG TGT GCA AAC CTT TCG AGA CTC CTC AAG CA - #T GAA AGG AAA AAC ATC     721    Glu Cys Ala Asn Leu Ser Arg Leu Leu Lys Hi - #s Glu Arg Lys Asn Ile    #       235    - TCC ATC TTT AAG CGA GAA CAG AAA GCA GCC AC - #C ACC CTG GGG ATC ATC     769    Ser Ile Phe Lys Arg Glu Gln Lys Ala Ala Th - #r Thr Leu Gly Ile Ile    #   250    - GTC GGG GCC TTT ACC GTG TGC TGG CTG CCA TT - #T TTC CTC CTC TCG ACA     817    Val Gly Ala Phe Thr Val Cys Trp Leu Pro Ph - #e Phe Leu Leu Ser Thr    255                 2 - #60                 2 - #65                 2 -    #70    - GCC AGA CCC TTC ATC TGT GGC ACT TCC TGC AG - #C TGC ATC CCA CTG TGG     865    Ala Arg Pro Phe Ile Cys Gly Thr Ser Cys Se - #r Cys Ile Pro Leu Trp    #               285    - GTG GAG AGG ACA TTT CTG TGG CTA GGC TAT GC - #A AAC TCT CTC ATT AAC     913    Val Glu Arg Thr Phe Leu Trp Leu Gly Tyr Al - #a Asn Ser Leu Ile Asn    #           300    - CCT TTT ATA TAT GCC TTC TTC AAC CGG GAC CT - #G AGG ACC ACC TAT CGC     961    Pro Phe Ile Tyr Ala Phe Phe Asn Arg Asp Le - #u Arg Thr Thr Tyr Arg    #       315    - AGC CTG CTC CAG TGC CAG TAC CGG AAT ATC AA - #C CGG AAG CTC TCA GCT    1009    Ser Leu Leu Gln Cys Gln Tyr Arg Asn Ile As - #n Arg Lys Leu Ser Ala    #   330    - GCA GGC ATG CAT GAA GCC CTG AAG CTT GCT GA - #G AGG CCA GAG AGA CCT    1057    Ala Gly Met His Glu Ala Leu Lys Leu Ala Gl - #u Arg Pro Glu Arg Pro    335                 3 - #40                 3 - #45                 3 -    #50    - GAG TTT GTG CTA CAA AAT GCT GAC TAC TGT AG - #A AAA AAA AGG TCA    TGATTCAT1112    Glu Phe Val Leu Gln Asn Ala Asp Tyr Cys Ar - #g Lys Lys Arg Ser    #               365    - TTGAAAGCAG AACAATGGAG ATGAAATAAA CAAGGCAAAA TAGAGGTGGA AA - #CAGAAGGA    1172    - AGTCATTTGC TGAGTCTGCA GAATGGAATG CAGCTTCTGT CCTTTCTTGG GA - #TGTCTAAA    1232    - ACATGACAAA CAGGGTGATC TGTTGTACAC ACTATCTTAT GAGGGAGATG GT - #GACTTCTC    1292    - CTTTTTTCTG TGGATCAGTG CTATTGTGTG TTCTCAGTTT AAGATAGCAG AT - #CATCTCAG    1352    - CAGTAAGCAC ACCAACAGAA CTGAGTTCCA GAAAGGAAGC AGTTTCTGGT GC - #TTTGCATA    1412    #          1417    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 365 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Gly Glu Lys Val Val Ile Gly Ser Ile Leu Th - #r Leu Ile Thr Leu Leu    #                 15    - Thr Ile Ala Gly Asn Cys Leu Val Val Ile Se - #r Val Cys Phe Val Lys    #             30    - Lys Leu Arg Gln Pro Ser Asn Tyr Leu Ile Va - #l Ser Leu Ala Leu Ala    #         45    - Asp Leu Ser Val Ala Val Ala Val Met Pro Ph - #e Val Ser Val Thr Asp    #     60    - Leu Ile Gly Gly Lys Trp Ile Phe Gly His Ph - #e Phe Cys Asn Val Phe    # 80    - Ile Ala Met Asp Val Met Cys Cys Thr Ala Se - #r Ile Met Thr Leu Cys    #                 95    - Val Ile Ser Ile Asp Arg Tyr Leu Gly Ile Th - #r Arg Pro Leu Thr Tyr    #           110    - Pro Val Arg Gln Asn Gly Lys Cys Met Ala Ly - #s Met Ile Leu Ser Val    #       125    - Trp Leu Leu Ser Ala Ser Ile Thr Leu Pro Pr - #o Leu Phe Gly Trp Ala    #   140    - Gln Asn Val Asn Asp Asp Lys Val Cys Leu Il - #e Ser Gln Asp Phe Gly    145                 1 - #50                 1 - #55                 1 -    #60    - Tyr Thr Ile Tyr Ser Thr Ala Val Ala Phe Ty - #r Ile Pro Met Ser Val    #               175    - Met Leu Phe Met Tyr Tyr Gln Ile Tyr Lys Al - #a Ala Arg Lys Ser Ala    #           190    - Ala Lys His Lys Phe Pro Gly Phe Pro Arg Va - #l Glu Pro Asp Ser Val    #       205    - Ile Ala Leu Asn Gly Ile Val Lys Leu Gln Ly - #s Glu Val Glu Glu Cys    #   220    - Ala Asn Leu Ser Arg Leu Leu Lys His Glu Ar - #g Lys Asn Ile Ser Ile    225                 2 - #30                 2 - #35                 2 -    #40    - Phe Lys Arg Glu Gln Lys Ala Ala Thr Thr Le - #u Gly Ile Ile Val Gly    #               255    - Ala Phe Thr Val Cys Trp Leu Pro Phe Phe Le - #u Leu Ser Thr Ala Arg    #           270    - Pro Phe Ile Cys Gly Thr Ser Cys Ser Cys Il - #e Pro Leu Trp Val Glu    #       285    - Arg Thr Phe Leu Trp Leu Gly Tyr Ala Asn Se - #r Leu Ile Asn Pro Phe    #   300    - Ile Tyr Ala Phe Phe Asn Arg Asp Leu Arg Th - #r Thr Tyr Arg Ser Leu    305                 3 - #10                 3 - #15                 3 -    #20    - Leu Gln Cys Gln Tyr Arg Asn Ile Asn Arg Ly - #s Leu Ser Ala Ala Gly    #               335    - Met His Glu Ala Leu Lys Leu Ala Glu Arg Pr - #o Glu Arg Pro Glu Phe    #           350    - Val Leu Gln Asn Ala Asp Tyr Cys Arg Lys Ly - #s Arg Ser    #       365    __________________________________________________________________________

We claim:
 1. Isolated mammalian serotonin receptor protein PCT-65, saidprotein being localized predominantly in the hippocampus, hypothalamusand spleen of a mammal, and exhibiting high affinity binding toserotonin and clozapine.
 2. The receptor protein of claim 1, whereinsaid protein is human PCT-65.
 3. A recombinant construct comprising apolynucleotide encoding the mammalian serotonin receptor of claim 1operably linked to a heterologous promoter.
 4. The recombinant constructof claim 3, wherein said polynucleotide is SEQ ID NO:
 6. 5. Aneukaryotic cell line in continuous culture expressing the PCT-65serotonin receptor protein of claim
 1. 6. The mammalian cell line ofclaim 5, wherein said PCT-65 serotonin receptor protein is encoded bySEQ ID NO:
 6. 7. The cell line of claim 5, wherein said cells arederived from a human.
 8. The cell line of claim 5, wherein said cellsare HEK
 293. 9. A method of screening a ligand for binding to the PCT-65serotonin receptor protein of claim 1, said method comprising the stepsof:transfecting a eukaryotic cell line with a gene encoding said PCT-65serotonin receptor protein in an expression vector; culturing saideukaryotic cell line to express said gene in media containing a ligandof said receptor; and measuring the binding of said ligand to saidreceptor produced by said eukaryotic cell line.
 10. The method of claim9, wherein said eukaryotic cell line is from a mammal.
 11. The method ofclaim 9, wherein said PCT-65 serotonin receptor is encoded by thepolynucleotide of SEQ ID NO: 6 or SEQ ID NO:
 8. 12. The method of claim9, wherein said eukaryotic cell line is HEK
 293. 13. The method of claim9, wherein said expression vector is pSRα-65.
 14. The method of claim 9,wherein said ligand is labeled.
 15. The method of claim 14, wherein saidlabel is radioactive.
 16. The method of claim 14, wherein said label iscolorimetric.
 17. A method of determining the ability of a drug toinhibit ligand binding to the PCT-65 serotonin receptor protein of claim1, the method comprising:transfecting a eukaryotic cell line with a DNAsequence encoding said PCT-65 serotonin receptor in an expressionvector; culturing said eukaryotic cell line to express said PCT-65receptor in media containing a ligand of said receptor; determining thelevel of binding of said ligand to said receptor; culturing the sameeukaryotic cell line to express said receptor in the presence of bothsaid ligand and said drug; and determining the level of binding of saidligand to said expressed receptor, wherein a lower level of binding inthe presence of said drug indicates that said drug is an inhibitor ofligand binding.
 18. The method of claim 17, wherein said cell line ismammalian.
 19. The method of claim 17, wherein said ligand is labeled.20. The method of claim 17, wherein said drug is labeled.
 21. The methodof claim 17, wherein said cells are HEK
 293. 22. The method of claim 17,wherein said expression vector is PSRα-65.
 23. The method of claim 17,wherein said ligand is serotonin.
 24. A method of determining theability of a drug to inhibit ligand binding to the PCT-65 serotoninreceptor protein of claim 1, the method comprising:transfecting aeukaryotic cell line with a DNA sequence encoding said PCT-65 serotoninreceptor in an expression vector; culturing said eukaryotic cell line toexpress said PCT-65 receptor in media containing a ligand of saidreceptor; isolating the membranes from said eukaryotic cell line,wherein the membranes contain the expressed PCT-65 receptor; determiningthe level of binding of said ligand to said receptor or said membranes;incubating said membranes in the presence of both said ligand and saiddrug; and determining the level of binding of said ligand to saidmembranes, wherein a lower level of binding in the presence of said drugindicates that said drug is an inhibitor of ligand binding.
 25. Thereceptor protein of claim 1, wherein said protein is rat PCT-65.
 26. Thereceptor protein of claim 25, wherein said rat PCT-65 is SEQ ID NO: 7.27. An isolated polynucleotide comprising the DNA sequence of SEQ ID NO:6 or SEQ ID NO: 8.